This invention relates generally to an oil-in-water emulsion, and more particularly, to an oil-in-water emulsion that functions as a blood-pool selective carrier or delivery vehicle for lipophilic imaging agents, or lipid-soluble derivatives of water- soluble, imaging agents incorporated therein.
Conventional water-soluble contrast media for x-ray computed tomography (CT) and magnetic resonance imaging (MRI) rapidly diffuse out of the blood following injection. Vascular imaging, for example, therefore depends on invasive intra-arterial infusion of large amounts of contrast media at or near the suspected site of disease. Despite administration of a bolus dose of contrast media, enhancement lasts for only a few seconds. In CT angiography, as a specific example, a large amount ( less than 200 ml) of a conventional water-soluble urographic agent is administered directly into the artery at a rate approaching 5 ml/sec. Such rapid administration can cause nausea and vomiting. Because conventional urographic agents are rapidly distributed throughout the vascular space before rapid renal elimination, CT scanning must be accomplished within 30 seconds of administration while the agent is still in the circulation phase. Intravascular contrast is rapidly lost as the agent diffuses into the extravascular space and distributes nonspecifically throughout the body. There is, therefore, a need for a delivery vehicle for CT scanning that can be administered less invasively and that will prolong the presence of the agent in the blood.
Several experimental CT agents have been developed to provide extended circulation time in the blood, including high molecular weight carboxymethyl dextrans and nanocrystalline particulates. Iodinated versions of the dextrans have opacified blood for up to 20 minutes, however, significantly delayed clearance (greater than a day) from the liver poses a concern. The nanocrystalline particulates comprising, in one example, solid ethyl diatrizoate having a particle size ranging from 200-400 nm, are also very slowly cleared by the reticuloendothelial system (RES) of the liver and spleen. There is, thus, a need for a delivery vehicle that will circulate in the blood for a prolonged period of time, but which will be metabolized and cleared from the system within an acceptable time period.
In addition to the foregoing experimental agents, several liposomal oil-in-water emulsions have been developed wherein the inclusion of polyethylene glycol (PEG) or a PEG derivative of a phospholipid, was found to reduce RES uptake and clearance of active agent from parenterally administered delivery active agent and to prolong the blood half life of the vehicles. Although liposomes and lipoproteins share some common structural lipid components and have considerable overlap in particle size, there remain significant differences in particle structure and in the mechanism of sequestration of the two particle types by their respective target tissues.
Liposomes, which are artificially prepared lipid vesicles formed by single or multiple polar lipid bilayers, consisting primarily of phospholipids and cholesterol, enclosing aqueous compartments are particulate in nature, and hence, have potential for delivering agents contained therein to the RES. Investigators have attempted to load liposomes with both ionic and non-ionic water-soluble urographic contrast media. However, stabilization of the resulting liposome against loss of contrast media from the bilayers has proven to be a major problem. Moreover, incorporation of neutral lipophilic agents into the bilayer is limited by the low capacity of the lipophilic agents to become incorporated in the membrane matrix and the restricted loading capacity of the liposome.
Lipoproteins, on the other hand, are naturally-occurring, oil-in-water emulsions composed of a monolayer of polar (amphiphilic) lipids that surround a neutral lipid core made up of cholesteryl esters and triglycerides. A variety of apolipoproteins associate with the polar monolayer of these lipid-transport particles. Each of the apolipoproteins plays a role as a recognition factor for tissue-selective, receptor-mediated uptake or in enzyme-mediated metabolism of the various classes of lipoproteins. Liposomes, which lack these specific surface recognition proteins, are rapidly sequestered by macrophages of the RES in the lungs, liver (Kupffer cells), spleen, and bone marrow. Liposomal biodistribution can be modulated somewhat by alteration of the surface charge, particle size, and chemical modification of surface components, although a significant portion of the modified liposomal material is still sequestered by the macrophages. A problem with RES-mediated particulates, such as the aforementioned liposomes is toxicity. Large imaging doses of particulate contrast agents have been associated with engorgement of the Kupffer cells of the liver resulting in sinusoidal congestion and consequent activation of macrophages which release toxic mediators.
Accordingly, there remains a great need in the art for less toxic delivery vehicles or compositions, including contrast-producing oil-in-water emulsions for diagnostic purposes that have prolonged blood circulation time, yet are cleared from the system within a reasonable period of time.
It is, therefore, an object of this invention to provide a delivery vehicle, specifically a blood-pool selective, surface-modified, oil-in-water emulsion, for transport of lipophilic agents, or lipophilic derivatives of water soluble agents, such as radiologic contrast agents.
It is another object of the invention to provide a blood-pool selective delivery vehicle, specifically a lipoprotein-like oil-in-water emulsion, that achieves prolonged retention in the circulation by avoiding sequestration by the RES.
It is still another object of this invention to provide a blood-pool selective delivery vehicle that is substantially free of liposomal contamination.
It is also an object of this invention to provide a delivery vehicle, specifically a blood-pool selective, surface-modified, oil-in-water emulsion, that remains in the blood for a prolonged period of time (on the order of 1 to 2 hours versus seconds) following intravenous administration (versus invasive arterial catheterization).
The foregoing and other objects are achieved by this invention which is a surface-modified synthetic oil-in-water lipid emulsion, resembling endogenous lipoproteins, in order to take advantage of the natural lipid transport system of a living being. The surface-modified oil-in-water emulsion of the present invention have been modified with derivatized polyethylene glycol or polyethylene glycol derivatives of phospholipids to prolong retention time in the blood, possibly by interfering with the association of the emulsion particles with apolipoproteins and/or opsonins which are responsible for mediating cellular uptake and circulatory elimination of the vehicle. Lipophilic agents or lipophilic derivatives of water-soluble agents which are diagnostically, therapeutically, or biologically active or inactive, inserted into the lipid core of the emulsion are retained in the blood.
In accordance with the present invention, the mean oil phase particle size is between 50 and 150 nm (number weighted), with a narrow size distribution (50 to 250 nm) wherein no more than 2% of the particles have a diameter that falls outside of the range (i.e., being greater than 250 nm). The emulsion should have no detectable particles with a diameter greater than 1 xcexcm. Moreover, the emulsion should not be contaminated with liposomes.
In a composition aspect of the invention, the synthetic oil-in-water emulsion of the present invention has the general formula:
1. up to 50% lipophilic core components (w/v);
2. up to 10% emulsifier (w/v);
3. up to 5% cholesterol (w/v);
4. up to 5% derivitized PEG or PEG-derivative of a phospholipid (w/v):
5. up to 5% osmolality adjusting agent (w/v);
6. optionally, up to 1% antioxidant (w/v); and
7. water to final volume.
The types of agents that can be administered by incorporation into the lipophilic core of the synthetic oil-in-water emulsions of the present invention are lipophilic contrast agents and/or lipophilic derivatives of conventional water-soluble contrast agents. The lipophilic core components comprise up to 50% (w/v) of the emulsion, and preferably between about 10% and 40% (w/v). The lipophilic core may comprise any pharmaceutically acceptable fat or oil of natural, synthetic, or semi-synthetic origin which is a pharmacologically inert nonpolar lipid that will locate in the lipophilic core of the oil-in-water emulsion. Specific examples include, without limitation, triglycerides, illustratively, triolein, a naturally-occurring triglyceride of high purity (available from a variety of commercial sources, such as Sigma Chemical Company, St. Louis, Mo.), or oils of animal or vegetable origin, such as soybean oil, safflower oil, cottonseed oil, canola oil, fish oils, and other biocompatible oils.
In preferred embodiments, the lipophilic core includes lipophilic contrast agents or lipophilic derivatives of water-soluble contrast agents that may be used for diagnostic purposes. For diagnostic purposes, exemplary agents include, but are not limited to, halogenated triglycerides, such as iodinated or fluorinated triglycerides; perfluorinated lower alkyls; or aliphatic esters of conventional water-soluble contrast agents, such as aliphatic esters of iopanoic acid, which agents may contain a stable or radioactive isotope of the halogen. The term xe2x80x9ccontrast agentxe2x80x9d or xe2x80x9cimaging agentxe2x80x9d is used herein to denote generically an agent useful for any imaging modality.
In particularly preferred embodiments, the lipophilic core includes a mixture of at least one pharmacologically inert (or inactive) oil and a contrast agent in a molar ratio in the range of 0.1 to 3. On a weight/weight (w/w) basis, the ratio of inert oil to contrast agent is from 0.1:1.0 to 2:1, and more preferably 1:1. Preferably, the lipophilicity of each core component is comparable to ensure suitable blending of the lipid components.
In iodinated embodiments, iodine-containing lipids, of the type known in the art, can be used. Such lipids include iodinated fatty acids in the form of glycerol or alkyl esters. However, in particularly preferred embodiments, the iodine-containing lipids are synthetic aromatic compounds of known purity that are stabilized against in vivo degradation of the iodine linkage. Illustrative examples of radioactive or non-radioactive halogenated triglycerides useful in the practice of the invention include, without limitation, iodinated triglycerides of the type described in U.S. Pat. No. 4,873,075 issued on Oct. 10, 1989; U.S. Pat. No. 4,957,729 issued on Sep. 18, 1990; and U.S. Pat. No. 5,093,043 issued on Mar. 3, 1992. Exemplary iodinated triglycerides are 2-oleoylglycerol-1,3-bis[7-(3-amino-2,4,6-triiodophenyl)heptanoate] (DHOG) and 2-oleoylglycerol-1,3-bis[4-(3-amino-2,4,6-triiodophenyl)butanoate] (DBOG), such as disclosed in International Publication No. WO 95/31181 published Dec. 14, 1995, the text of which is incorporated herein by reference.
Clinically, 123I, 125I, and 131I are the iodine isotopes most often used with currently available scanning instrumentation. Of course, 131I-radiolabeled triglycerides may be used for therapeutic purposes, as is known in the art. However, any radioactive isotope of iodine is within the contemplation of the invention. A listing of all iodine isotopes is available, for example, at pages Misc. 47-49 of the Merck Index, 11th Edition, and at pages 11-68 to 11-70 of the Handbook of Chemistry and Physics, 72d Edition, CRC Press, 1991-1992. It should be noted that 127I is the naturally-occurring stable isotope and is not considered to be xe2x80x9cradioactivexe2x80x9d.
In fluorinated embodiments, specific examples include stable (19F) or radioactive (18F) fluorinated triglycerides that are analogous to the iodinated triglycerides discussed above, illustratively glyceryl-2-oleoyl-1,3-bis(trifluoromethyl)phenyl acetate. In alternative embodiments of the invention, fluorine-containing lipids may be esters or triglycerides of perfluoro-t-butyl-containing fatty acid compounds, such as described in U.S. Pat. Nos. 5,116,599 and 5,234,680, illustratively, 7,7,7-trifluoro-6,6-bis (trifluoromethyl)-heptanoic acid or 8,8,8-trifluoro-7,7-bis(trifluoromethyl)-octanoic acid. Other examples include the perfluorinated low molecular weight hydrocarbons, useful as ultrasound imaging agents, such as described in U.S. Pat. No. 5,716,597.
In still further embodiments of the invention, the contrast agent may comprise brominated compounds, such as brominated ethyl esters of fatty acids or monobrominated perfluorocarbons. Of course, these examples are merely illustrative of the many specific examples of lipophilic compounds suitable for use in the practice of the invention, and are not in any way intended to be exclusive or limiting.
While the invention is described in terms of the delivery of diagnostic contrast agents, it is to be understood that therapeutic agents, specifically radiopharmaceuticals, may be included in the lipophilic core of the synthetic oil-in-water emulsion of the present invention.
The monolayer surrounding the nonpolar lipophilic core comprises up to about 10% (w/v) of an amphipathic lipid monolayer component, which may be an emulsifier. Phospholipids of natural, synthetic, or semi-synthetic origin are suitable for use in the practice of the invention. Traditional lipid emulsions for delivery of contrast agents use natural phospholipids, such as soy lecithin and egg phosphatidylcholine (e.g., Intralipid). In preferred embodiments of the present invention, the emulsion components are synthetic, semi-synthetic, and/or naturally occurring components of known origin, purity and relative concentrations. The improper use of egg lecithins (mixtures of phospho-lipids) and/or crude oils (cottonseed, poppy seed, and the like), as in typical prior art emulsions, may result in variable and non-reproducible compositions.
In a specific advantageous embodiment, dioleoylphosphatidylcholine (DOPC) is used as an emulsifier, or monolayer surfactant. DOPC is a semi-synthetic, chemically defined phospholipid emulsifier of high purity (available from Avanti Polar Lipids, Alabaster, Ala.). Of course, other surface active agents that are suitable for parenteral use can be substituted for all or a portion of the polar lipid monolayer component. The naturally-occurring phospholipids are advantageous because these phospholipids are biocompatible and have an appropriate phase transition temperature, i.e., they are in the liquid state at physiologic temperatures.
In addition to the foregoing, polyethylene glycol-linked lipids are incorporated into the monolayer. A derivatized polyethylene glycol, such as methoxy polyethylene glycol (MPEG), having a molecular weight between about 1000 and 6000 and/or polyethylene glycol-derivatized lipids, such as MPEG-linked to phosphatidylethanolamine or distearoyl phosphatidylethanolamine are preferred. The PEG component should comprise between about 0.1 and 30 mole percent of the monolayer components for achieving attenuation of retention time of the delivered contrast agent in the blood.
In preferred embodiments, the synthetic MPEG-linked phospholipids may contain fatty acyl groups, including but not limited to myristoyl, palmitoyl, steroyl, oleoyl, or combinations thereof. MPEGs can be covalently linked to the phospholipid moiety by succinate, carbamate, or amide linkages, or by other covalent linkages known to those skilled in the art. MPEG-linked phospholipids are available commercially from Matreya, Inc., Pleasant Gap, Pa. and Avanti Polar Lipids, Inc., Alabaster, Ala. Preferred MPEG-modified phospholipids include MPEG-linked phosphatidylethanolamine; MPEG-2000-1,2-distearoyl; and MPEG-2000-1,2-dioleoyl phosphatidylethanolamine. Of course, other polysaccharides can be associated with phospholipids, or other suitable membrane lipid moieties, to modify the surface of the monolayer in order to block association of the emulsion particles with apolipoproteins and/or opsonins, thereby interfering with receptor-mediated uptake and prolonging the residence time of the lipid emulsion in the blood.
The composition also contains a sterol, which is preferably cholesterol, in an amount of up to 5% by weight in order to stabilize the emulsion, and preferably in the range of 0.4 to 0.5% (w/v). In accordance with preferred embodiments of the invention, the molar ratio of sterol to emulsifier, which may be a natural, synthetic, or semi-synthetic phospholipid, has been found to directly affect the particle diameter and dimensional stability. The preferred molar ratio of sterol to phospholipid for achieving an emulsion of the desired size in the range of 0.05 to 0.70, and more specifically at 0.40 for delivery of iodinated triglycerides.
The remainder of the emulsion formulation comprises the bulk or aqueous phase containing up to 5% (w/v) USP glycerol as an osmolality adjusting agent. In the practice of a preferred embodiment of the present invention, the aqueous phase is de-ionized water of a grade suitable for parenteral administration. The inclusion of salt (NaCl), such as by the use of 0.9% saline, or ionic buffers, in the aqueous phase results in unstable emulsions that have a mean particle diameter as much as twice the size of salt-free emulsions. Furthermore, the presence of salt in the formulation has an adverse effect on the ability of the emulsion to survive autoclave sterilization without a significant change in mean particle size as well as on the temporal stability of an autoclaved emulsion. Any ionic species in the formulation adversely impacts the long term stability of the emulsion.
Other conventional additives, such as antioxidants, buffers, preservatives, viscosity adjusting agents, and the like, may be included in the composition. In particular, up to 1% w/v of an antioxidant, such as xcex1-tocopherol, flavinoids, BHT, or BHA, is recommended. However, the additive should not adversely affect the physical and biological characteristics of the emulsion, such as particle size, shelf stability, and biodistribution.
The techniques used to formulate the oil-in-water emulsions of the present invention are important in achieving small particle diameter, uniform size distribution, lack of liposome contamination, etc.
In accordance with a method of making aspect of the invention, the lipophilic components of the oil-in-water emulsion including nonpolar core lipids, polar lipid emulsifiers, and other lipophilic components, such as contrast agents, are blended together to form a premixed lipid phase. The aqueous components are combined and added to the premixed lipid phase. The premixed lipid phase and aqueous components are homogenized to form a crude oil-in-water emulsion. The crude oil-in-water emulsion is subjected to ultra high energy emulsification to produce a fine oil-in-water emulsion having a mean particle diameter of the oil phase between 50 to 150 nm with greater than 98% of the particles being less than 250 nm. In preferred embodiments of the invention, the fine oil-in-water emulsion is sequentially filtered.
In a preferred method aspect of the invention, the lipid components are initially blended or homogenized with USP glycerol using a high speed mixer, such as a Polytron homogenizer (Kinematica GmbH, Lucerne, Switzerland) or Ultra Turrax (IKA-Works, Cincinnati, Ohio), operating at 12,500 rpm under a nitrogen atmosphere at 55xc2x0 C. for at least 5 minutes. Then, the aqueous components are added to the anhydrous glycerol-lipid emulsion and emulsified by high speed mixing or homogenization at 25,000 rpm under the same, or similar, conditions to form a crude oil-in-water emulsion. Final processing is accomplished with ultra high energy mixing equipment, such as a MicroFluidizer high pressure homogenizer (Model 110S, Microfluidics Corp., Newton, Mass.; see, U.S. Pat. No. 4,533,254), or equivalent equipment, such as the Emulsiflex (Avestin Inc., Ottawa, Ontario, Canada) or the Manton-Gaulin (APV Gaulin Rannie, St. Paul, Minn.), operating in the recycling mode at 35-60xc2x0 C. and 10,000 to 30,000 psi, and preferably at about 14,700-23,000 psi, for up to about 20 minutes. After processing, the emulsion is passed sequentially through sterile 0.45 xcexcm and 0.22 xcexcm sterile filters. The sequential filtration removes any large particles and offers the potential of end-point sterilization of the product.
The temperature for high energy mixing is illustrative, and should be chosen relative to the contrast agent. In other words, the temperature should be greater than or equal to the phase transition temperature or melting point of the contrast agent or emulsifier (phospholipid or MPEG-linked phospholipid). An upper bound, however, is determined by whether the temperature would cause degradation or decomposition of any components in the composition.
The use of an ultra high pressure homogenizer ensures small particle size with a narrow size range distribution. Conventional systems for forming emulsions, such as homogenizers, sonicators, mills, and shaking systems provide a shearing force on the liquid components whereas the ultra high energy mixing equipment puts the emulsion components under pressure and forces them through small openings to reduce particle size. Size distribution may be measured by submicron laser photon correlation spectroscopy (PCS) on a Nicomp 370 Dynamic Laser Light Scattering Autocorrelator (Nicomp Particle Sizing Systems, Santa Barbara, Calif.) or similar equipment. A lipid emulsion, which is suitable for the practice of the present invention, will have a mean particle diameter less than about 250 nm, and preferably in the range of 50 to 150 nm as measured by Nicomp number weighting analysis. The particles should have a narrow size distribution, with about 98% of the particles being in the 50 to 250 nm. No particles should be detected with a diameter of greater than 1 xcexcm.
In a method of use aspect of the invention, an oil-in-water emulsion of the present invention containing a contrast enhancing agent is administered to a mammal and the mammal is subjected to x-ray computed tomographic imaging after the emulsion has reached stable blood levels, e.g., 1-30 minutes post-injection and prior to decline in levels (up to about 2 hours). In alternative methods of use, appropriate oil-in-water emulsions, containing contrast agents suitable for other diagnostic modalities, such as proton magnetic resonance imaging (MRI), 19F-MRI, ultrasonography, or scintigraphy may be administered for visualization and/or detection.